Buffers are widely used for pH control of chemical processes. A buffered solution resists changes in pH when acids or bases are added or when dilution occurs. Biochemists are particularly concerned with buffers because the proper functioning of any biological system depends on pH. The rate of one particular enzyme-catalyzed reaction varies with pH. For an organism to survive, pH of each subcellular compartment has to be precisely controlled so that each reaction proceeds at the proper rate. The majority of biological samples that are used in research are made in buffers, such as phosphate, Tris-HCl at pH around 7.
Microbial fuel cells (MFCs), which can directly generate electricity from biodegradable substance, have rapidly gained increasing research attention. Microbes oxidize organic substrates to supply electrons to the anode; the electrons then travel through an external circuit to the cathode and participate in reduction reactions. Associated with these reactions is the generation of H+ and OH− from water electrolysis in the analyte and catholyte, respectively, which creates a pH imbalance in system. Since the pH imbalance produces a 0.059 V/pH potential loss, minimizing the pH imbalance is necessary for maximizing the power densities. Phosphate buffers are usually used to stabilize the pH and reduce the internal resistance, which in turn enhances the system performance. In order to have a sufficient supply of protons in a cathode compartment, a high concentration of buffer is needed, and over time it might still become depleted. A stable supply of fresh buffer will be attractive.
Buffers are of wide utility in analytical chemistry as well. Reversed-phase high performance liquid chromatography (RP-HPLC) has always been a very powerful tool for organic compound analysis, especially biological sample analysis. Buffers like phosphate or acetate are widely employed in the mobile phase in the analysis of ionizable compounds. Because the retention of ionizable acid/base compounds has a strong dependence of their degree of ionization, a correctly chosen buffer pH will ensure that the ionizable functional group is in a single form, whether ionic or neutral. When developing a rugged method, it is desirable to select a mobile phase with a final pH at least one pH unit away from any analyte's pK value to cause ionization or suppression of the analytes. Slight variations in pH can have a dramatic impact on separation, in terms of selectivity, capacity factor, peak shape, resolution and reproducibility. An improper pH for ionizable analytes often leads to asymmetric peaks that are broad, tail, split or shoulder. And sharp, symmetrical peaks are necessary in quantitative analysis in order to achieve low detection limits, low relative standard deviations between injections, and reproducible retention times. Even in the analysis of non-ionizable compounds, it is often equally important to control pH when working with field samples in the presence of ionizable contaminants or impurities so as to eliminate the interference of undesirable peaks. If the sample solution is at a pH damaging to the column, the buffer will quickly bring the pH of the injected solution to a less harmful pH. Another technique of RP-HPLC, ion-pair chromatography (IPC) also requires precise control of pH in the mobile phase, because variation of pH in the mobile phase can introduce large changes in the degree of ionization of not only the solutes but also the ion-pairing reagent. In practice, a chromatographer usually measure the pH of the buffer additives before mixing it with other solvents, but the pKa values of the acids used to prepare the buffers change with the solvent composition (and each in a different degree), and so does the pH of the buffer. Sometimes, the pH is measured after mixing the buffer with the organic modifier; even in this instance, the potentiometric system is usually calibrated with aqueous standards, and the measured pH is not the true pH of the mobile phase. Theoretically, a chromatographer can estimate the concentration of buffer needed to achieve a desired pH for separation based on calculation from equations. However, in reality pH can vary significantly from those calculations. Therefore the analyst has to experimentally determine and report the value for the mobile phase pH with a calibrated pH meter to ensure reproducibility results. When developing a method, the analyst might have to adjust the mobile phase many times before it reaches the optimum condition. With one stock buffer solution, the final concentration of buffer can only be varied by varying the ratio of buffer to the organic solvent, which largely limits the flexibility of the optimization process. To make different buffer stock solutions is tedious and time-consuming. If the buffer concentration can be varied simply by varying the applied current, it will provide a much efficient way and reduce lots of waste solvents.
pH-based separation of proteins with ion-exchange chromatography is another chromatographic technique that relies on buffers. Initially it employs nearly linear pH gradients generated from mobile phase (ampholyte buffers) and stationary phase (weak anion-exchange column) to elute proteins in the order of their pI, and was termed “chromatofocusing”. More recently, the technique was developed into “gradient chromatofocusing”, which employs common buffers with low molecular mass instead of polymeric ampholytes. There are two types of gradients; a pH gradient in time at column outlet causing differential elution of proteins and a pH gradient in distance within the column affecting the focusing of the protein bands. HPLC gradient pumps are typically used to generate the linear pH gradient in time by varying the ratio of high-pH buffer and low-pH buffer, which are mixtures of buffer components with pKa values approximately equally spaced throughout the gradient pH range.
Capillary electrophoresis (CE) is another powerful separation tool for analysis of proteins and peptides, as well as drug enantiomers. Its unprecedented resolution allowing separation of species with very subtle differences in structure is a consequence of its extremely high efficiency, which, to some extent, depends on the running buffers it employs. Manipulation of buffer pH is usually a key strategy to optimize a separation, since buffer pH not only determines the extent of ionization of each individual analyte, but also strongly influences the charge of the capillary wall surface and the zeta potential, consequently affects both electrophoretic and electroosmotic velocities. Electrolysis of water is one of the most significant reactions occurring at the inlet and outlet vials in a CE experiment, the resulting H+ and OH− can change the pH in the vials. Thus, to successfully maintain the pH of the buffer, large vials should be used and the buffer must have adequate buffering capacity to neutralize the H+ and OH− produced, and the buffer vials should be replenished regularly. By manipulating the running buffers, sample pre-concentration can also be achieved to overcome the drawback of limited sensitivity in CE.
Essentially a buffer is a mixture of an acid and its conjugate base. There must be comparable amounts of the conjugate acid and base (say, within a factor of 10) to exert significant buffering. The most common way to prepare a buffer solution is to decide the ratio of the conjugate acid-base pair based on Henderson-Hasselbalch equation, and weigh out the two components separately to obtain the desired ratio and then dissolve in water. An alternative is to weigh out one of the component, and produce the other component by a specified amount of strong acid or strong base to yield the desired ratio. Although it is a common practice to adjust pH of certain buffer solution with concentrated strong acid or base, it is easy to overshoot by adding too much of the titrants and have to make another solution and start over again.